Cell surface antigen detection method

ABSTRACT

The present invention provides a method for assaying a sample of cells for the presence of a cell surface antigen. More particularly, the present invention describes methods for detecting the presence of plasma and cell bound autoantibodies against cell surface antigens. In addition, the present invention provides a method of crossmatching donor platelets and transfusion recipients.

This invention was made with the support of the Government of the UnitedStates of America. The government may have certain rights in theinvention.

This is a continuation of copending U.S. application Ser. No. 124,905filed on Nov. 24, 1987, now U.S. Pat. No. 5,176,998, which is acontinuation-in-part of copending U.S. application Ser. No. 936,125filed on Dec. 1, 1986 now U.S. Pat. No. 4,810,632.

DESCRIPTION

1. Technical Field

The present invention relates to immunological methods for determiningplatelet histocompatibility type and for characterizing theanti-platelet immune status of an individual.

2. Background

The platelet histocompatibility type and anti-platelet immune status ofan individual both play important roles in the management of clinicalconditions due to anti-platelet immune response. Anti-platelet immuneresponses resulting in the production of platelet-destroying antibodiesoccur as a result of, among other things, pregnancy, platelettransfusion therapy and anti-platelet autoimmune disease.

Of particular interest to the present invention is platelet transfusiontherapy. Since its beginning over three-quarters of a century ago,platelet transfusion therapy has played an increasingly important rolein the supportive care of patients with bone marrow failure. However,one impediment to progress in this field has been the finding that asignificant proportion of recipients become refractory to repeatedtransfusions from random donors. Platelet rejection appears to be due inmany cases to anti-platelet antibodies in the transfusion recipient(donee) induced as a result of alloimmunization.

Alloimmunization is the process wherein an individual producesantibodies in response to exposure to an alloantigen. An alloantigen isan antigen, typically a protein, that exists in alternative (allelic)forms in different individuals of the same species, and thus induces animmune response when one form is transferred (as by transfusion ortissue graft) to members of the same species who have not previouslybeen exposed to it. Class I human leukocyte antigens (HLA) are one groupof alloantigens that are found on the surface of platelets and arecapable of inducing alloantibodies (antibodies against alloantigens)that mediate platelet rejection in transfusion recipients. Yankee et al.first demonstrated that transfusions of HLA-matched platelets fromsingle donors frequently results in good increments in platelet count intransfusion recipients. That is, crossmatching of donor platelet andrecipient HLA type has been found to reduce platelet rejection intransfusion recipients.

Unfortunately, HLA matched platelet donors are available for only aminority of alloimmunized patients, and the response of recipients topartially matched donor platelets is less predictable. The recognitionof cross-reacting groups and the differential expression of certain HLAantigens (e.g., B12) on platelets has facilitated educated guessingduring donor selection. However, a means of selecting donor plateletswithout resorting to trial and error would be advantageous.

Characterization of an individual's anti-platelet immune status is, aspreviously mentioned, also important in managing autoimmune diseases.One such disease is chronic immune thrombocytopenic purpura (ITP), asyndrome of destructive thrombocytopenia due to an antibody against aplatelet-associated antigen (McMillan, N. Engl. J. Med. 304:1135-1147(1981), and Kelton et al Semin. Thromb. Haemost., 8:83-104 (1982). vanLeeuwen et al., (Blood, 59:23-26 (1982)) first provided evidence thatautoantibodies were present in some ITP patients. They noted that of 42antibody eluates from ITP platelets, 32 would bind to normal but not tothrombasthenic platelets; the remaining eluates bound to both. Sincethrombasthenic platelets are deficient in platelet glycoproteins (GP)IIb and IIIa, they suggested that these ITP patients had autoantibodiesto one of these glycoproteins.

Direct evidence for anti-glycoprotein autoantibodies in chronic ITP hasbeen provided by subsequent studies using a variety of methods. Woods etal. showed binding of autoantibodies from ITP patients to the GPIIb/IIIacomplex or to GPIb attached to microtiter wells with monoclonalantibodies and confirmed these observations by immunoprecipitation. SeeWoods et al., Blood, 63:368-375 (1984) and Woods et al., Blood,64:156-160 (1984). Using the former method, they noted anti-GPIIb/IIIaor anti-GPIb autoantibodies in about 10% of patients, much less than thepercentage observed by the indirect studies of van Leeuven et al. supra.

Other investigators also detected antiplatelet autoantibodies in chronicITP patients using immunoblotting (Mason et al., Br. J. Haematol.,56:529-534 (1984) and Beardsley et al., J. Clin. Invest. 74:1701-1707(1984)), immunoprecipitation, (Woods et al., Blood, 63:368-375 (1984),Woods et al., Blood, 64:156-160 (1984) and Devine et al., Blood,64:1240-1245 (1984)), inhibition of murine monoclonal anti-GPIIb/IIIaantibody binding to ITP platelets (Varon et al., Proc. Natl. Acad. Sci.USA, 80:6992-6995 (1983)) and crossed immunoelectrophoresis (Szatkowskiet al., Blood, 67:310-315 (1986)). Nugent et al. ("Proceedings of theINSERM Symposium on utilization of monoclonal antibodies for theunderstanding and detection of platelet activity." Amsterdam, ElsevierScience Publishers, 1986) and Asano et al., (Blood, 66:1254-1260 (1985))have established human hybridomas from ITP lymphocytes which synthesizemonoclonal antiplatelet antibodies. Some of these are specific forplatelet glycoproteins (Nugent et al., supra).

Of the assays used for demonstrating antiglycoprotein autoantibodies inchronic ITP, the microtiter well assay (Woods et al., Blood, 63:368-375(1984) and Woods et al., Blood, 64:156-160 (1984)) is most easilyadaptable to clinical use. However, the low percentage of positive tests(about 10%) when compared to that of van Leeuwen et al. (about 76%)suggested that solubilization of the platelets prior to antibodysensitization may alter some of the epitopes. For this reason, an assay(immunobead assay) for antiglycoprotein autoantibodies was designedwhere platelets are sensitized prior to their solubilization to takeadvantage of the possibility that the epitopes expressed by the plateletsurface antigens may remain more stable when bound to antibody. Thisassay can measure both platelet-associated and plasma autoantibodies.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a method of assaying a sample of cellsfor the presence of a cell surface antigen, which method comprises thesteps of:

(a) admixing said cells with an antibody capable of immunoreacting withsaid antigen to form a cell-sensitizing reaction admixture,

(b) maintaining said cell-sensitizing reaction admixture for a timeperiod sufficient to form an immunocomplex containing said antigen andsaid antibody, thereby forming sensitized cells,

(c) lysing said sensitized cells to provide an aqueous composition, saidcomposition containing solubilized immunocomplex when said immunocomplexis formed in step (b),

(d) admixing said aqueous composition with a solid support capable ofspecifically binding said immunocomplex, thereby forming a solid/liquidphase admixture,

(e) maintaining said solid/liquid phase admixture for a time periodsufficient for any of said immunocomplex present to bind to said solidsupport, thereby forming a solid-phase bound complex, and

(f) assaying for the presence of any bound complex that formed in step(e).

The present invention also provides a method for detecting the presenceof a cell surface antigen, which method comprises the steps of:

(a) providing an aliquot of cells to be assayed, any cells of saidaliquot bearing the surface antigen to be detected having animmunocomplex containing an antibody to said antigen immunoreacted withsaid antigen;

(b) lysing the cells of said aliquot to provide cellular debris, andwhen said immunocomplex was present in the aliquot, a solublizedimmunocomplex; and

(c) assaying from the presence of said solubilized immunocomplex.

In another embodiment, the present invention contemplates a method ofassaying platelet compatibility between donor and patient, which methodcomprises the steps of:

(a) admixing an aliquot of serum or plasma from a patient with plateletsof a prospective donor to form an aqueous, liquid admixture;

(b) maintaining said admixture under biological assay conditions for apredetermined time period sufficient for any alloantibodies present inthe serum or plasma to immunoreact with a platelet antigen to form animmunocommplex;

(c) lysing the platelets of said admixture to form cellular debris and asolublized immunocomplex when said alloantibodies and said plateletantigen were present in said admixture; and

(d) assaying for the presence of said immunocomplex.

Also contemplated by the present invention is a method of typing cellsfor the presence of a group of cell surface antigens, which methodcomprises the steps of:

(a) reacting an aliquot of a cell sample to be typed with an antibodythat reacts with an antigen epitope common to substantially all of thegroup of the cell surface antigens sought to form an immunocomplex;

(b) lysing the cells of said sample to provide cellular debris and asolublized immunocomplex;

(c) reacting the solubilized imanunocomplex with indicating paratopicmolecules that immunoreact with at least one specific cell surfaceantigen epitope of the group of cell surface antigens to form a secondimmunocomplex; and

(d) assaying for the presence of said second immunocomplex.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

The term "antibody" in its various grammatical forms is used herein as acollective noun that refers to a population of immunoglobulin moleculesand/or immunologically active portions of immunoglobulin molecules,i.e., molecules that contain an antibody combining site or paratope.

An "antibody combining site" is that structural portion of an antibodymolecule comprised of heavy and light chain variable and hypervariableregions that specifically binds antigen.

The phrase "antibody molecule" in its various grammatical forms as usedherein contemplates both an intact immunoglobulin molecule and animmunologically active portion of an immunoglobulin molecule.

Exemplary antibody molecules are intact immunoglobulin molecules,substantially intact immunoglobulin molecules and those portions of animmunoglobulin molecule that contain the paratope, including thoseportions known in the art as Fab, Fab' F(ab')₂ and F(v).

Fab and F(ab')₂ portions of antibodies are prepared by the proteolyticreaction of papain and pepsin, respectively, on substantially intactantibodies by methods that are well known. See for example, U.S. Pat.No. 4,342,566 to Theofilopolous and Dixon. Fab' antibody portions arealso well known and are produced from F(ab')₂ portions followed byreduction of the disulfide bonds linking the two heavy chain portions aswith mercaptoethanol, and followed by alkylation of the resultingprotein mercaptan with a reagent such as iodoacetamide. An antibodycontaining intact antibody molecules are preferred, and are utilized asillustrative herein.

The phrase "monoclonal antibody." in its various grammatical formsrefers to a population of one species of antibody molecule of determined(known) antigen-specificity. A monoclonal antibody contains only onespecies of antibody combining site capable of immunoreacting with aparticular antigen and thus typically displays a single binding affinityfor that antigen. A monoclonal antibody may therefore contain abispecific antibody molecule having two antibody combining sites, eachinununospecific for a different antigen.

As used herein, the term "biological assay conditions" is used for thoseconditions wherein a molecule useful in this invention such as anantibody binds to another useful molecule such as an antigen epitopewithin a pH value range of about 5 to about 9, at ionic strengths suchas that of distilled water to that of about one molar sodium chloride,and at temperatures of about 4 degrees C. to about 45 degrees C.

The word "complex" as used herein refers to the product of a specificbinding agent-ligand reaction. An exemplary complex is an immunoreactionproduct formed by an antibody-antigen reaction.

A "specific binding agent" is a molecular entity capable of selectivelybinding another molecular entity or ligand. Exemplary specific bindingagents are paratopic molecules, complement proteins or fragmentsthereof, S. aureus protein A, and the like. Preferably, the specificbinding agent binds its ligand when the ligand is present as part of acomplex.

II. Assay Methods

The present invention broadly contemplates a method of detecting thepresence of a cell surface antigen. In accordance with this method,cells to be assayed, typically blood cells, are admixed with an antibodycapable of immunoreacting with the antigen while it is present on thecell surface. The resulting admixture is a first immunoreactionadmixture and it is typically referred to as a cell-sensitizing reactionadmixture because it produces "antibody-sensitized" cells.

Cell surface antigens known to be clinically useful markers whosepresence can be determined by a method of the present invention includethose that determine histocompatibility, such as a majorhistocompatibility complex (MHC) antigen, particularly a human leukocyteantigen (HLA). Useful cell surface antigens also include glycoproteins(GP) that comprise membrane associated receptors that bindArg-Gly-Asp-containing proteins. Such glycoproteins include GPIb, GPIIb,GPIIIa and the cell surface complexes containing those proteins, such asthe GPIIb/IIIa complex.

Antibodies capable of immunoreacting with antigens on the surface ofcells are well known and include those that are available from theAmerican Type Culture Collection (ATCC), Rockville, Md.

The cell-sensitizing reaction admixture is maintained for a time periodsufficient for an immunocomplex containing the antibody and the cellsurface antigen to form on the cellular surface. As is well known, thetime period required for immunocomplex formation depends on a variety offactors including temperature and concentration of reactants. Typically,the time period is predetermined for a given set of reaction conditionsby well known methods prior to performing the assay. Under biologicalassay conditions, the maintenance time period is usually from minutes tohours such as 30 minutes to about 2 hours.

Maintaining the cell-sensitizing reaction for a time sufficient forimmunocomplex formation results in the production of antibody-sensitizedcells which are typically separated by washing and centrifugation fromany non-immunoreacted antibody. If the cells have the cell surfaceantigen being assayed for, the sensitized cells will have a cell surfaceassociated immunocomplex.

To determine the presence of any cell surface immunocomplex formed, thesensitized cells are lysed to produce an aqueous composition containingsolubilized cell surface protein. Lysing can be accomplished by admixingthe sensitized cells with an aqueous buffer such as water or saline anda surfactant capable of solubilizing cell surface protein. Surfactantscapable of solubilizing cell surface proteins include detergents such aspolyoxyethylene (9) octyl phenyl ether, available under the trademarkTriton X-100 from Rohm and Hass Co., Inc., Philadelphia, Pa.

The lysing reaction admixture formed upon admixture of the cells andsurfactant is maintained for a time period sufficient for the surfactantto solubilize (dissociate from the cell membrane) surface protein of thecells, including any cell surface immunocomplex formed. Typically, thetime period used for solubilization is predetermined and usually is inthe range of from minutes to hours such as about 15 minutes to about 2hours.

Methods for detecting solubilization of cell surface protein are wellknown and include using immunological methods to monitor the aqueousphase of the lysing reaction admixture for appearance of a protein knownto be associated with the surface of the cells being assayed. Forinstance, the glycoproteins IIb and IIIa are found associated with thesurface of all normal platelets and can be used as immunological markersof surface protein solubilization when platelets are being assayed.

The aqueous solubilized protein composition formed after maintenance ofthe lysing reaction admixture usually contains cellular debris as wellas solubilized protein. In preferred embodiments, the solubilizedprotein is separated from the cellular debris, typically bycentrifugation.

To detect the presence of any solubilized surface immunocomplex formed,a portion of the aqueous solubilized protein composition is admixed witha solid support capable of specifically binding the immunocomplex,thereby forming a solid/liquid phase (capturing reaction) admixture.

Solid supports capable of specifically binding a solubilized surfaceimmunocomplex are typically comprised of a specific binding agentaffixed (operatively linked) to a solid matrix. Preferably, the solidphase-affixed specific binding agent is an antibody molecule capable ofimmunoreacting with the solubilized surface immunocomplex. It should benoted that an antibody capable of immunoreacting with either the antigenor the antibody of the surface immunocomplex can be used.

Useful solid matrices are well known in the art. Such materials arewater insoluble and include the cross-linked dextran available under thetrademark SEPHADEX from Pharmdcia, Piscataway, N.J.; agarose;polyvinylchloride, polystyrene, cross-linked polyacrylamide,nitrocellulose or nylon-based webs such as sheets, strips or paddles; ortubes, plates or the wells of a microtiter plate such as those made frompolystyrene, polycarbonate or polyvinylchloride.

When present as part of a solid support, a specific binding agent istypically affixed to a solid matrix by adsorption from an aqueousmedium, although other modes of affixation, such as covalent coupling,well known to those skilled in the art, can be used.

The solid/liquid phase admixture is maintained for a time periodsufficient for any solubilized surface immunocomplex present to be boundby the solid support and thereby form a solid-phase bound complex.Preferably, the maintained solid support is then separated from anynon-bound protein, typically by washing.

Assaying the maintained solid support for the presence of solid-phasebound complex thereby provides a means for detecting the presence of thesurface antigen of interest in the cell sample. Such assaying istypically performed by forming a labeling reaction admixture by admixingthe maintained solid support with a labeled specific binding agentcapable of specifically binding the immunocomplex portion of thesolid-phase complex. Preferably, the labeled specific binding is capableof binding either the antigen or the antibody of the surfaceimmunocomplex when the surface immunocomplex is itself bound to thesolid support.

The labeling reaction admixture is maintained for a time periodsufficient for the labeled specific binding agent to react with(specifically bind) any solid-phase immunocomplex present and therebyform a labeled solid-phase product. Any non-bound labeled specificbinding agent is then typically separated from the solid support.

The presence of any labeled solid-phase product formed is thendetermined by known assay procedures that depend, as is well known, onthe type of label used. The presence of labeled solid-phase productindicates the presence of the surface antigen of interest in the cellsample.

As used herein, the terta "solubilized immunocomplex" means a complexformed of an antibody bound to a cell surface antigen epitope whosepresence is sought that is formed by reacting the antibody and cellsurface antigen epitope followed by lysis of the cell to form cellulardebris and a solubilized immunocomplex. The immunocomplex can be presentin the cell sample analyzed as obtained from a patient (donor) as wherethe cell sample is a platelet sample from a patient with immunethrombocytopenic purpura (ITP) that contains autoantibodies to aplatelet antigen such as the glycoprotein IIb/IIIa complex orglycoprotein Ib. The immunocomplex can also be formed by immunoreactionof alloantibodies of a patient with platelets of other cells of aprospective donor. The immunocomplex can still further be formed byimmunoreaction of antibodies to a major histocompatibility complex (MHC)antigen epitope such as an HLA class I or class II antigen epitopepresent on the surface of the cell.

Another assay method for detecting the presence of a cell surfaceantigen comprises the steps of providing an aliquot of cells to beassayed. The cells of such an aliquot that bear the surface antigen tobe detected having an immunocomplex that contains an antibody to theantigen sought immunoreacted with the antigen. Here, again, theimmunocomplex, if present, can be present in the sample as obtained fromthe patient (donor) or can be prepared after obtaining the cell sample.The cells of the aliquot are lysed to provide cellular debris, and whenthe immunocomplex was present in the aliquot, to form a solubilizedimmunocomplex. The presence of the solubilized immunocomplex isthereafter assayed for.

In preferred practice, the cellular debris formed in thelysis/solubilization step is separated from the solubilizedimmunocomplex by any convenient means such as centrifugation. Theseparated, solubilized immunocomplex is thereafter affixed to a solidphase support to form a solid phase-affixed immunocomplex. Theaffixation can be by physical adsorption, immunoreaction using solidphase-bound antibodies directed to the antibodies of the immunocomplex,like monoclonal mouse anti-human antibodies as are available from theAmerican Type Culture Collection of Rockville, MD (ATCC) under theaccession number HB43, or by binding with S. aureus protein A coated onthe solid support. The immunocomplex is thereafter assayed for as asolid phase-affixed immunocomplex.

In one embodiment, the cells of the sample are platelets. In such anembodiment, the antibodies of the immunocomplex can be autoantibodiesfrom the sample donor (patient) and those autoantibodies can be directedto the glycoprotein IIb/IIIa complex or glycoprotein Ib. In anotherembodiment, the platelets or other cells are from a first person (donor)and the antibodies of the immunocomplex can be alloantibodies from asecond person (donee), as where cross-matching of platelets or othercells from a prospective donor, and serum or plasma from a patient(donee) provides the antibodies of the immunocomplex. The antibodies ofthe immunocomplex can also be directed to a group of cell surfaceantigens such as a tissue rejection-related antigen like an antigen ofthe MHC or an HLA antigen. Antibodies directed to an epitope present onsubstantially all HLA antigens as well as those directed to individualHLA-specific antigen epitopes are available from the ATCC as well asfrom the antiserum bank at the National Institutes of Health (NIH),Bethesda, Md.

Thus, a method is also provided for assaying platelet or other cellularcompatibility between a donor and a patient (donee). In this method, analiquot of serum or plasma from a patient suspected of havingalloantibodies to a donor platelet or other cellular antigen is admixedwith platelets or other cells of the donor to form an aqueous, liquidadmixture. The admixture so formed is maintained under biological assayconditions for a predetermined time period sufficient for anyalloantibodies present in the serum or plasma to immunoreact with theplatelet or other cellular antigen to form an immunocomplex. Theplatelets or other cells of the admixture are lysed to form cellulardebris and a solubilized immunocomplex, when the alloantibodies andplatelet or other cellular antigen were present in the admixture. Thepresence of the immunocomplex is thereafter assayed for. If theimmunocomplex is not found to be present, the donor and donee arecompatible, and vice versa.

The present invention also provides a method for typing cells for thepresence of various surface antigens such as HLA antigens. Here analiquot of a cell sample to be typed is reacted with an antibody thatreacts with an antigen epitope common to substantially all of the groupof cell surface antigens to be assayed for such a monoclonal antibodythat reacts with the backbone of the HLA antigen to form animmunocomplex. The cells are thereafter lysed to provide cellular debrisand a solubilized immunocomplex. The solubilized immunocomplex isthereafter reacted with indicating paratopic molecules; i.e., wholeantibodies or portions of antibodies that contain the paratope, thatimmunoreact with at least one member of the antigen group such as anHLA-specific antigen epitope to form a second immunocomplex. Thepresence of the second immunocomplex is then assayed for. Where HLAantigens are sought, the first-named antibodies and indicating paratopicmolecules are available from the ATCC and the NIH.

Where a complete profile of cell surface antigens is desired, as whereHLA typing is carried out, it is often convenient to divide thesolubilized immunocomplex into a plurality of aliquots. The aliquots arethereafter individually reacted with members of a panel of indicatingparatopic molecules, and the presence of second immunocomplex from eachof panel members as assayed for.

The assay methods of the present invention utilize a solid supportcapable of specifically binding a solubilized immunocomplex. Usefulsolid supports are typically comprised of a specific binding agentaffixed (operatively linked) to a solid matrix.

A specific binding agent can be linked to a label or indicating meansand used to form a labeled immmunoreaction product.

The terms "indicating means" and "label" are used herein to includesingle atoms and molecules that are capable of producing a detectablesignal and of being linked to an antibody or used separately.

The indicating means can be a fluorescent labeling agent that chemicallybinds to antibodies or antigens without denaturing them to form afluorochrome (dye) that is a useful immunofluorescent tracer. Suitablefluorescent labeling agents are fluorochromes such as fluoresceinisocyanate (FIC), fluorescein isothiocyanate (FITC),5-dimethylamin-1-naphthalenesulfonyl chloride (DANSC),tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200sulphonyl chloride (RB 200 SC) and the like. A description ofimmunofluorescence analysis techniques is found in Marchalonis et al.,"Immunofluorescence Analysis" 189-231, supra, which is incorporatedherein by reference.

In preferred embodiments, the indicating group is an enzyme, such ashorseradish peroxidase (HRP), glucose oxidase, or the like. Where theprincipal indicating group is an enzyme such as HRP or glucose oxidase,additional reagents are required to visualize the fact that a complex(immunoreaction product) has formed. Such additional reagents for HRPinclude hydrogen peroxide and an oxidation dye precursor such asdiaminobenzidine. An additional reagent useful with glucose oxidase is2,2'-azino-di-(3-ethyl-benzthiazoline-G-sulfonic acid) (ABTS).

Radioactive elements are also useful labeling agents and are usedillustratively herein.

An exemplary radiolabeling agent is a radioactive element that producesgamma ray emissions. Elements which themselves emit gamma rays, such as¹²⁴ I, ¹²⁵ I, ¹²⁸ I, ¹³¹ I, ¹³² I and ⁵¹ Cr represent one class of gammaray emission-producing radioactive element indicating groups.Particularly preferred is ¹²⁵ I. Another group of useful indicatinggroups are those elements such as ¹¹ C, ¹⁸ F, ¹⁵ O and ¹³ N whichthemselves emit positrons. The positrons so emitted produce gamma raysupon encounters with electrons present in the animal's body. Also usefulis a beta emitter, such as ¹¹¹ indium.

The labeling of proteinaceous specific binding agents is well known inthe art. For instance, antibodies produced by hybridomas can be labeledby metabolic incorporation of isotope containing amino acids provided asa component in the tissue culture medium. See for example Galfre et al.,Meth. Enzymol., 73:3-46 (1981). The techniques of protein conjugation orcoupling through activated functional groups are particularlyapplicable. See, for example, Aurameas, et al., Scand. J. Immunol., Vol.8, Suppl. 7:7-23 (1978) and U.S. Pat. No. 4,493,795 which isincorporated herein by reference. Methods for conjugating enzymes toproteins may be found in U.S. Pat. No. 3,791,932 and U.S. Pat. No.3,839,153. In addition, site directed coupling reaction can be carriedout so that the label does not substantially interfere with theimmunoreaction of the second receptor with apo B-100. See, for example,Rodwell et al., Biotech., 3:889-894 (1985).

EXAMPLES

The following examples are intended to illustrate, but not limit, thepresent invention.

1. Characterization of the Antiplatelet Immune Status of AutoimmuneIndividuals A. Study Subjects

I studied plasma or platelet samples obtained between Jan. 1, 1982 andJul. 1, 1986 from 44 patients with chronic ITP, 20 control subjects and20 thrombocytopenic controls (acute non-lymphocytic leukemia, 3patients; acute lymphoblastic leukemia, 2 patients; bone marrowtransplantation patients with anti-HLA antibodies, 3 patients; aplasticanemia, 2 patients; non-Hodgkins lymphoma, 3 patients (one withcryoglobulins); cirrhosis, one patient; myeloproliferative disease, 3patients; and carcinoma on chemotherapy, 3 patients). Patients with ITPwere thrombocytopenic with normal or increased numbers of megakaryocytesand without evidence of other types of immune thrombocytopenia.

B. Assays for Antiglycoprotein Autoantibody

The specificity of both the immunobead and microtiter assays isdetermined by the monoclonal antiglycoprotein antibody employed. Thefollowing murine monoclonal antibodies were used: anti-GPIIb/IIIa-2A9,3F5, 2G12 (provided by Dr. V. L. Woods) - 2A9 is specific for GPIIb andthe others are complex-specific; anti-GPIb-P3 (provided by Drs. ZaverioRuggeri and Theodore Zimmerman, Scripps Clinic); and antihuman IgG(American Type Culture Collection, Rockville, Md., ATCC HB-43).Monoclonal antibodies (50 ug) were labeled with 500 uCi of ¹²⁵ I usingthe chloramine-T method. All incubations in both assays were carried outat room temperature.

It is known that in detergent extracts GPIIb and GPIIIa form a complexand that GPIb is complexed with platelet glycoproteins IX and V.Therefore, as measured by these two assays, anti-GPIIb/IIIaautoantibodies could be against epitopes on either GPIIb or GPIIIa andanti-GPIb specific for GPIb, GP V or GP IX. However, for the purposes ofthis report, results will be reported as either anti-GPIIb/IIIa oranti-GPIb referring to autoantibodies against proteins of the GPIIb/IIIacomplex or the GPIb complex.

C. Immunobead Assay

This assay can be used to measure either platelet-associatedautoantibody or plasma autoantibody.

(i) Immunobead Preparation

Anti-IgG-coated immunobeads were prepared by incubating polystyrenebeads (Poly-Sep, Polysciences Inc., Warrington, Pa.) with murinemonoclonal antihuman IgG (ATCC HB-43) in saline for 60 min at abead/antibody ratio of 2000:1 by weight (e.g., 100 mg of beads to 50 ugof anti-IgG in 2 ml of saline). The beads were then centrifuged for 10sec at maximum speed in a tabletop centrifuge (International ClinicalCentrifuge, Model 65133M). After washing once with 10 ml of 0.05%Tween-20 in phosphate-buffered saline, pH 7.4 (PBS-Tween), nonspecificbinding sites are blocked by incubation of the beads in 2% bovine serumalbumin (BSA) in PBS-Tween for 60 min followed by four washes inPBS-Tween.

(ii) Platelet Preparation

Platelets from EDTA-anticoagulated blood were obtained from the patientor from a normal donor and washed six times with 0.05M isotonic citratebuffer. To prepare antibody-sensitized platelets, washed normalplatelets (10⁸ in 0.1 ml) were incubated with 1.0 ml of patient orcontrol plasma, containing PGE₁ (1 ug/ml) and theophylline (1 uM), for60 min at room temperature and then washed four times with 0.05 Mcitrate buffer containing PGE₁ and theophylline. Patient platelets (10⁸)or the antibody-sensitized platelets were resuspended in 900 ul ofcitrate buffer containing leupeptin (100 ug/ml) and then solubilized byadding 100 ul of 10% Triton X-100. Control samples were handledsimilarly.

(iii) Assay

The solubilized platelets from each sample were centrifuged at 12,000 xgfor 5 min. Preliminary studies showed that this step was required,particularly in anti-GPIb autoantibody studies, to prevent falselyelevated values. The supernate is then incubated for 60 min with 100 mgof anti-IgG-coated immunobeads to allow attachment of IgG and any boundantigen. After four washes with PBS-Tween, the presence of specificantigen is demonstrated by incubating the beads with 1.0 ml of PBS-Tweencontaining about 400,000 cpm of ¹²⁵ I monoclonal antibody specific foreither anti-GPIIb/IIIa (a cocktail of 3 monoclonal antibodies specificfor noncompeting sites, 2A9, 3F5, and 2G12) or anti-GPIb (P3) for 60 minat room temperature and then washing four times with PBS-Tween. Thebeads were resuspended in 1 ml of buffer and 0.5 ml was removed fordetermining radioactivity. Data are expressed as a binding ratio of cpmof patient sample/mean cpm of three control samples. The mean percentvariation results of replicate samples of control platelets andplatelets sensitized with control plasma were: anti-GPIIb/IIIa-10.1±7.5(14 studies) and 9.1±8.1 (31 studies), respectively, and anti-GPIb-6.8±7.0 (17 studies) and 7.9±7.6 (24 studies), respectively. Patientsamples with a binding ratio of >1.3 are considered positive (>2 S. D.over control). Preliminary studies show that positive reactivity can beremoved by adsorption of plasma with excess platelets. In addition,storage of samples for up to four days at 4° C. did not affect plateletor plasma control values.

D. Microtiter Well Assay

Details have been previously published (Woods et al., Blood, 63:368-375(1984) and Woods et al., Blood, 64:156-160 (1984)). Briefly, washedplatelets (10⁹ ml) or CEM leukemic cells (10⁷ ml) in PBS containingleupeptin (100 ug/ml) were solubilized in 1% Triton X-100 for 30 min at4° C. and then ultracentrifuged (100,000 xg for 60 min). The lysates arestored at -70° C. Microtiter wells are coated overnight at 5° C. with100 ul of either anti-GPIIb/IIIa (2A9 or 3F5) or anti-GPIb (P3) at aconcentration of 5 ug/ml. After six washes with 200 ul of PBS-Tween, theremaining binding sites were blocked for 50 min with 200 ul of 2% BSA inPBS-Tween. After six washes with PBS-Tween, 100 ul of platelet lysate orthe antigen negative CEM lysate, diluted 1:10, were added and incubatedfor 50 min. This allows attachment of the specific platelet antigen tothe well-bound monoclonal antibody. After six washes, appropriatedilutions (1:10 for screening plasma and higher dilutions if positive)of patient or control plasma were added and incubated for 50 min. Aftersix washes, 100 ul of radiolabeled murine monoclonal antihuman IgG(about 100,000 cpm) were added and after 50 min incubation and six finalwashes, the radioactivity of each well was determined. The percentvariation for replicate control plasmas is -5.0±7.7 for anti-GPIIb/IIIaand -1.9±6.4 for anti-GPIb (Woods et al., Blood, 63:368-375 (1984) andWoods et al., Blood, 64:156-160 (1984)). Samples with a percent increaseof >11 were considered positive (>2 S. D.).

E. Results

(i) Chronic ITP Patients

The results were divided into two groups for evaluation: (1)pre-splenectomy patients--the initial study sample was obtained prior tosplenectomy although in some of these patients additional studies werealso performed after surgery; (2) posts-plenectomy patients--the patientwas first studied after surgery.

(ii) Pre-Splenectomy Studies (Table 1)

Twenty-six patients were studied; 16 subsequently had their spleensremoved and eight entered a complete remission, six were splenectomyfailures and two were lost to follow up. Platelet-associatedautoantibody was measured using the immunobead assay in seven of the 26patients. Of these, six (85.7%) were positive with ratios ranging from4.9 to 30.1 (control valued <1.3); five had anti-GPIIb/IIIa and one hadanti-GPIb autoantibodies.

                                      TABLE I                                     __________________________________________________________________________    ANTI-PLATELET GLYCOPROTEIN AUTOANTIBODIES                                     PRE-SPLENECTOMY CHRONIC ITP PATIENTS                                          Platelet             Anti-GPIIb/IIIa                                                                             Anti-GPIb                                  Patient                                                                            Count           Immunobead    Immunobead                                 Number                                                                             (per mm.sup.3)                                                                      Splx                                                                             Resp                                                                             PAIgG                                                                             P-Assoc                                                                            Plasma                                                                            Well P-Assoc                                                                            Plasma                                                                            Well                              __________________________________________________________________________     1    2000 Yes                                                                              ?  --  16.7 2.3 Neg  --   0.8 Neg                                2    9000 Yes                                                                              CR --  --   0.8 Neg  --   1.0 Neg                                3    9000 Yes                                                                              CR --  --   0.9 Neg  --   0.9 Neg                                4    9000 Yes                                                                              NR --  --   4.5 Neg  --   0.9 Neg                                5   10000 No -- --  --   0.9 Pos(320)                                                                           --   1.0 Pos(80)                            6   14000 Yes                                                                              NR --  --   1.5 Neg  --   1.1 Neg                                7   18000 Yes                                                                              CR 12962                                                                             --   0.6 Neg  --   1.0 Neg                                8   19000 No -- 4423                                                                              --   1.4 Neg  --   0.9 Neg                                9   20000 No -- 4873                                                                              --   1.3 Neg  --   0.9 Neg                               10   23000 Yes                                                                              CR --  --   1.7 Neg  --   --  Neg                               11   34000 Yes                                                                              CR 7353                                                                              --   1.6 Neg  --   --  Neg                               12   39000 No -- 30990                                                                             --   0.9 Neg  --   2.0 Neg                               13   40000 Yes                                                                              NR 3453                                                                              --   1.0 Neg  --   0.9 Neg                               14   40000 Yes                                                                              NR --  30.1 5.5 Neg  --   1.1 Neg                               15   42000 No -- --   0.6 0.7 Neg  1.0  1.2 Neg                               16   46000 No -- 9036                                                                              --   1.1 Neg  --   0.8 Neg                               17   50000 Yes                                                                              NR 11049                                                                             --   0.9 Neg  --   0.9 Neg                               18   50000 Yes                                                                              NR 17143                                                                             16.1 3.9 Neg  --   0.8 Neg                               19   50000 No -- --  10.0 2.5 Neg  --   0.8 Neg                               20   69000 No -- 7286                                                                               0.7 0.6 Neg  4.9  1.8 Neg                               21   85000 Yes                                                                              CR 1351                                                                              --   1.2 Pos(640)                                                                           --   --  Neg                               22   100000                                                                              No -- --  --   0.9 Neg  --   1.2 Neg                               23   106000                                                                              Yes                                                                              CR 5152                                                                              16.2 1.4 Neg  --   --  Neg                               24   112000                                                                              No -- 3537                                                                              --   0.9 Neg  --   3.6 Pos(40)                           25   145000                                                                              Yes                                                                              ?  3692                                                                              --   3.1 Neg  --   1.1 Neg                               26   26100 Yes                                                                              CR 3075                                                                              --   0.8 Neg  --   1.2 Neg                               __________________________________________________________________________     Splx  splenecotomy:                                                           Resp  response;                                                               CR  Normalization of platelet count;                                          NR  no response or relapse after an initial response or ? if lost to          followup;                                                                     PAIgG  plateletassociated IgG (ng/10.sup.9 plts);                             Immunobead  results of plateletassociated (PAssoc) or plasma immunobead       assay expressed as a ratio of patient/control results (positive > 1.3);       well  results of microtiter well assay expressed as negative (Neg) or         positive (Pos) with titers of positive tests shown in parenthesis.       

Platelet-associated autoantibodies in three of these patients (patients14, 18 and 23) were also studied after splenectomy (Table 2). In thepatient who attained a complete remission (patient 23), theanti-GPIIb/IIIa autoantibodies present prior to splenectomy were nolonger demonstrable after surgery. In the two others, the autoantibodiespersisted despite splenectomy and although both obtained a temporaryincrease in the platelet count after surgery (135,000/mm³ in one and313,000/mm³ in the other), they both became severely thrombocytopenic(<20,000) within a few weeks after surgery. Table 2 also compares theplatelet-associated IgG (PAIgG) and the platelet-associatedanti-GPIIb/IIIa values prior to and after splenectomy. In patient 23,who attained a complete remission, and patient 14, who responded onlypartially, the values of both determinations varied concordantly.However, in patient 18 whose platelet count became normal briefly aftersurgery but who later relapsed (platelet count 14,000/mm³), the PAIgGvalues became normal in concert with the platelet count but theplatelet-associated anti-GPIIb/IIIa remained elevated.

Plasma autoantibodies were studies in all patients using both assays;16/26 (61.5%) were positive in at least one of the assays. Eleven hadcirculating anti-GPIIb/IIia and three had anti-GPIb autoantibodies whenstudied using the immunobead assay; ratios varied from 1.4 to 5.5. Ofthese patients with positive immunobead assays, only one (patient 24)was also positive using the microtiter assay. Two patients with negativeimmunobead assays (patients 5 and 21) were positive using the microtiterassay; one of these (patient 5) had antibody in the microtiter assay toboth GPIIb/IIIa and GPIb.

                  TABLE 2                                                         ______________________________________                                        THE EFFECT OF SPLENECTOMY ON ANTI-GPIIIb/IIIa                                 AUTOANTIBODY AND PLATELET-ASSOCIATED IgG                                                              Platelet                                                                              Anti-   PAIgG                                       Sample*           Count   GPIIb/IIIa                                                                            (ng per                               Patient                                                                             Day      Therapy  (per mm.sup.3)                                                                        (Ratio) 10.sup.9 plts)                        ______________________________________                                        14    -25      P         40,000 28.8    8,900                                        0       P         96,000 21.2    5,847                                       +5       --       135,000 25.4    6,595                                       +52      --       109,000 31.1    19,500                                18    -111     --        51,000 --      17,140                                       0       P        118,000 16.1    4,400                                       +5       --       103.000 13.7    4,394                                       +8       --       313,000 11.3    2,611                                 23    -1       P        106,000 16.2    5,152                                       +28      --       465,000  1.0      985                                 Con-                    180-400,000                                                                           <1.3    <3,300                                trol                                                                          Values                                                                        ______________________________________                                         Anti-GPIIb/IIIa  plateletassociated autoantibodies against the platelet       glycoprotein IIb/IIIa complex measured by the immunobead assay;               PAIgG  plateletassociated IgG;                                                P  high dose prednisone;                                                      *Number of days before (-) or after splenectomy; day 0 is the day of          surgery.                                                                 

(iii) Post-Splenectomy Studies (Table 3)

All patients in this group failed to maintain a normal platelet countafter splenectomy. Platelet-associated autoantibody was measured usingthe inununobead assay in four of the 18 patients in this group. Threewere positive, one with autoantibodies against GPIIb/IIIa and the othertwo against GPIb.

Plasma autoantibodies were demonstrable using the immunobead assay inten of the 18 patients (55%), seven with anti-GPIIb/IIIa and three withanti-GPIb. Seven of the ten patients with positive immunobead assaysalso had autoantibodies of the same specificity shown by the microtiterassay. Two patients (patients 9 and 12) were positive in the microtiterassay but negative by the immunobead method. One patient (patient 4) hadautoantibodies against both antigens.

(iv) Thrombocytopenic Controls

Platelets (eight patients) and plasma (20 patients) from patients withnon-immune thrombocytopenia were studied using both assays. Negativeresults were obtained in every instance using both the immunobead andthe microtiter assays (data not shown).

                                      TABLE 3                                     __________________________________________________________________________    ANTI-PLATELET GLYCOPROTEIN AUTOANTIBODIES                                     POST-SPLENECTOMMY CHRONIC ITP PATIENTS                                        Platelet      Anti-GPIIb/IIIa                                                                             Anti-GPIb                                         Count         Immunobead    Immunobead                                        Patient                                                                           (per mm.sup.3)                                                                      PAIgG                                                                             P-Assoc                                                                            Plasma                                                                            Well P-Assoc                                                                            Plasma                                                                            Well                                     __________________________________________________________________________    27  1000  --  --   --  Neg  --    3.3*                                                                              Pos(6400)                               28  3000  --  --   2.2 Neg  --   ( ) Neg                                      29  3000   6911                                                                             --   1.0 Neg  --   1.0 Neg                                      30  3000  59800                                                                             --   1.3 Pos(40)                                                                            --   2.6 Pos(80)                                  31  6000  --  7.0**                                                                              9.4 Pos(640)                                                                           --   1.2 Neg                                      32  6000  --  --   0.9 Neg  -    0.9 Neg                                      33  7000  53000                                                                             --   5.2 Pos(320)                                                                           --   0.9 Neg                                      34  7000  --  --   1.2 Neg  17.2 2.0 Pos(20)                                  35  9000  --  --   0.6 Neg  1.0  0.9 Pos(40)                                  36  9000  --  --   1.0 Neg  --   1.1 Neg                                      37  13000 --  --   2.5 Neg  --   0.9 Neg                                      38  14000 --  0.6  0.7 Neg  5.7  1.3 Pos(80)                                  39  21000 42396                                                                             --   3.8 Neg  --   0.8 Neg                                      40  24000  6326                                                                             --   0.9 Neg  --   1.1 Neg                                      41  34000 --  --   0.6 Neg  --   1.0 Neg                                      42  40000  5650                                                                             --   2.6 Neg  --   1.1 Neg                                      43  42000 19024                                                                             --   1.0 Neg  --   0.9 Neg                                      44  117000                                                                              11795                                                                             --   2.1 Pos(40)                                                                            --   1.1 Neg                                      __________________________________________________________________________     PAIgG  plateletassociated IgG (ng/10.sup.9 plts);                             immunobead  results of plateletassociated (PAssoc) or plasma immunobead       assay expressed as a ration of patient/control results (positive > 1.3);      well  results of the microtiter well assay expressed as negative (neg) or     positive (pos) with titers of positive tests shown in parenthesis.            *0.1 ml of plasma used to sensitize platelets.                                **0.18 × 10.sup.8 platelets assayed.                               

F. Discussion

The above results illustrate experience with two methods for measuringantiplatelet autoantibodies in chronic ITP: the immunobead assay whichis capable of measuring both platelet-associated and plasmaautoantibodies and the microtiter assay useful in detecting only plasmaantibodies.

Platelet-associated autoantibodies were noted in nine of 11 ITP patients(81.8%; seven with anti-GPIIb/IIIa and two with anti-GPIb). Although thenumber of patients studied is too small to evaluate statistically,positive tests were seen in about the same frequency in thrombocytopenicpatients studied first pre-or post-spenectomy.

Plasma autoantibodies were noted in a lesser frequency; 28/44 werepositive (63.6%). Using the immunobead assay, 24/44 patients haddemonstrable circulating autoantibody (18 with anti-GPIIb/IIIa and sixwith anti-BPIb). Fewer positive samples were noted using the microtiterassay as reported previously (Woods et al., Blood, 63:368-375 (1984) andWoods et al., Blood, 64:156-160 (1984)). Only 12/44 patients werepositive using this assay (five with anti-GPIIb/IIIa, five withanti-GPIb and two with both). Three patients had autoantibodiesdemonstrable only with this method. This assay was positive in almostone-half of the post-spenectomy patient group (all who were refractory)while only three of the 26 patients in the pre-operative group werepositive. Whether this assay predicts more severe disease or reflectsthe tendency of our laboratory to receive samples from more severelyaffected patients is not known.

These autoantibody assays have distinct advantages over assays forplatelet-associated IgG (PAIgG). First, they allow the directdemonstration of either platelet-associated or plasma autoantibodiesagainst defined platelet proteins confirming the autoimmune nature ofthe patient's disease. Conversely, the PAIgG assay measures IgG onplatelets of unknown specificity. The adaptation of the PAIgG assay usedby van Leeuwen et al., Blood, 59:23-26 (1982), where the relativebinding to normal and thrombasthenic platelets is used can providesimilar although indirect information in patients with anti-GPIIb/IIiaantibodies but the method is cumbersome and thrombasthenic platelets arenot available to more laboratories. It is of interest that my rate ofanti-GPIIb/IIIa positivity, using the combination of assays, is quitesimilar to theirs. Second, autoantibody test results in patients withnon-immune thrombocytopenia have thus far been completely negative withthese two assays while PAIgG results are positive in many patients witha variety of diagnoses (Mueller-Eckhardt et al., Br. J. Haematol.,52:49-58 (1982) and Kelton et al., Blood, 60:1050-1053 (1982)). Althoughit seems likely, as suggested by the studies of Kelton et al. (supra),that positive PAIgG results may reflect immune-mediated plateletdestruction, it must be acknowledged that an increase in PAIgG does notnecessarily show the presence of autoantibody. A divergence betweenanti-GPIIb/IIIa and PAIgG results noted in one of our ITP patients(patient 18) supports this contention. Her autoantibody values remainedelevated after splenectomy despite normalization of both the plateletcount and the PAIgG results. This patient subsequently relapsed,suggesting that this assay may be useful in predicting the ultimateoutcome of splenectomy. Obviously these data are preliminary. Althoughautoantibodies were not seen in the group of thrombocytopenic controlpatients reported here, it seems likely that when patients with collagenvascular disease or lymphoma are screened using these assays thatpositive results maybe seen in some of these patient groups since theyhave been shown to have other types of autoantibodies (e.g., anti-RBCantibodies).

The difference in the results between the immunobead and microtiter wellassays is of interest. It is known from the study of purified proteinantigens that there are two types of epitopes: sequential andtopographic (Berzofsky, Science, 229:932-940 (1985)). Seqpaentialepitopes involve amino acid sequences of one section of the proteinwhile topographic epitopes involve regions of the molecule remote insequence but close in three dimensional space due to the tertiarymolecular structure. The most likely explanation for the greater percentpositivity with the inununobead assay is that solubilization of theplatelets prior to incubation with antibody, which is required for themicrotiter assay, in some way disturbs the antigenic epitope whileincubation of antibody and platelets prior to solubilization stabilizesit. I postulate that the microtiter assay measures sequential or stabletopographic epitopes while the immunobead assay measures unstabletopographic epitopes as well. Preliminary studies in my laboratorycomparing the ability of plasma autoantibodies to precipitate theGPIIb/IIIa complex after incubation of plasma with surface-labeledplatelets to that of precipitating radiolabeled purified GPIIb/IIIasupport this hypothesis. Plasmas positive in both the microtiter andimmunobead assays (three studied) are capable of precipitating both thepurified GPIIb/IIIa complex and the complex from surface-labeledplatelets while plasmas positive in the immunobead but negative in themicrotiter assay (four studied) are able to precipitate only the complexfrom surface-labeled platelets. Obviously, further studies will beneeded to confirm these preliminary findings.

These assays are both adaptable to the measurement of another as yetunidentified autoantibodies when appropriate monoclonal antibodiesbecome available. Since many patients with chronic ITP have nodemonstrable autoantibody, it seems likely that autoantibodies againstother platelet-associated antigens (e.g., phospholipids, glycolipids,etc.) are present.

In summary, the present studies describe the use of two assays for themeasurement of autoantibodies to specific platelet proteins. Resultsshow that the majority of patients with chronic ITP have autoantibodiesagainst either the platelet GPIIb/IIIa complex or against GPIb.Differences in the frequency of positive results seen in the two assaysprovide evidence for epitopes which have varying degrees of stabilityupon solubilization.

2. Antiplatelet Immune Status Characterization in Platelet TransfusionPatients A. Patient and Donor Population

Fifty one transfusion episodes involving 7 patients with bone marrowfailure were studied (Table 4). All were refractory to random donorplatelets prior to entry on study. Transfusion episodes associated withfever, infection or spenomegaly were not routinely excluded if goodincrements from any donor were seen within 24 hours. No patientdemonstrated evidence of DIC during the study period. Patient 1 wasdropped from the study when he became refractory to all donors a fewdays before his death, associated with ARDS and mechanical ventilation.Patient 2 was dropped from the study when he became persistently febrileand refractory to all donors. He was subsequently determined to have adisseminated fungal infection. Donors were selected from family memberswhen possible (n-16), otherwise from paid community volunteers (n-35).HLA matching was as follows: A-9; B,B2X-5; C-19; D-8; unknown,related-7, unknown, unrelated-3.

                  TABLE 4                                                         ______________________________________                                        PATIENT CHARACTERISTICS                                                                                            Number of                                                            Evaluable                                                                              Different                                Patient                                                                             Age/Sex  Diagnosis    Transfusions                                                                           Donors                                   ______________________________________                                        1     42/M     AA, allo-BMT 22       14                                       2     32/M     ALL, auto-BMT                                                                              2        2                                        3     21/M     AML          5        3                                        4     36/F     AML          2        2                                        5     41/M     AML          1        1                                        6     71/M     AML          17       11                                       7     57/M     2°MDS->AML                                                                          2        2                                                       Totals       51       35                                       ______________________________________                                         AA  aplastic anemia;                                                          alloBMT  allogenic bone marrow transplantation;                               autoBMT  autologous bone marrow transplantation;                              AML  acute myelogenous leukemia;                                              MDS  myelodysplastic syndrome.                                           

B. Assessment of Transfusion Outcome

Each transfusion episode was evaluated by computing the 1 hour correctedcount increment (CCI) from the following standard formula: ##EQU1## Whenavailable, a later CCI (8 to 24 hours) was also calculated. A successfuloutcome was defined as a 1 hour CCI of >7500. In one instance, a 1 hourCCI of 7289 with a 6 hour CCI of 4252 was considered "successful" when atransfusion from an HLA matched sibling given less than 24 hours latergave a 1 hour CCI of 8391 and 6 hour CCI of 2670. In 2 instances where 1hour post platelet counts were not available, an 8 hour CCI <0 notassociated with overt nonimmunologic causes of poor platelet survivalwas considered as evidence of an unsuccessful transfusion outcome.

C. Murine Monoclonal Antibodies

HB-43 is an IgG kappa antibody derived from the 1410K67 cell line, whichrecognizes the Fc portion of human IgG. HB-95 is an IgG₂ antibodyderived from the W6/32 cell line, which recognizes a monomorphic epitopeon the heavy chain of human HLA A, B and C antigens. The intactheterodimer is required for full expression of the epitope, althoughHB-95 binds weakly to isolated heavy chains. Both cell lines wereobtained from the American Type Culture Collection (Rockville, Md.) andwere used to produce ascites in Pristane-primed BALB/c mice, from whichantibody was purified by protein A affinity chromatography.

¹²⁵ I labeling was accomplished by the chloramine-T procedure.

D. Preparation and Storage of Platelets

(i) Normal Platelets

Blood from normal adult volunteers was drawn into EDTA and platelet-richplasma separated by centrifugation at 400 g for 10 minutes. Theplatelets were pelleted by centrifugation at 1000 g for 15 minutes,washed 4 times with 1.0 ml of 0.05 M citrate buffer (14.7 g sodiumcitrate, 5.9 g sodium chloride, 25.0 g dextrose in 1,000 ml distilled H₂O; adjusted to pH 6.2 with 0.05 M citric acid), and resuspended incitrate buffer to approximately 10⁹ /ml. Platelets were used fresh orstored in suspension at 4° C. for 24-48 hours.

(ii) Donor Platelets

Approximately 3 ml of platelet concentrate was obtained at the end ofeach phoresis procedure. This was divided into 1 ml aliquots andcentrifuged at 750 g for 90 seconds to remove red cells. The plateletswere centrifuged at 3000 g for 5 minutes and washed 4 times. Theplatelet concentrate could be kept for 24-48 hours at 4° C. prior towashing without affecting assay results.

(iii) Platelet Storage

To assess the effect of storage on the immunobead assay, 1.0 ml plateletaliquots (1.3×10⁹) from 2 normal donors were stored both in suspensionat 4° C. and by freezing in liquid nitrogen, then tested at intervals of1, 6, 15, 30 and 60 days against 2 control plasmas and 2 plasmas knownto contain high titer anti-HLA antibodies. Platelets to be frozen weresuspended in autologous plasma with 5% dimethylsulfoxide in plastictubes, frozen at -70° C. overnight and then kept in liquid nitrogen.After thawing in a 37° C. waterbath, they were washed twice, resuspendedin approximately 1 ml of citrate buffer and counted. Parallel aliquotswere suspended in normal saline with 0.02% sodium azide in plastic tubesand kept at 4° C. Prior to assay, the platelets were resuspended bypipetting, washed twice and counted.

E. Preparation and Storage of Plasma and Serum

All assays were conducted using plasma, except as noted. Plasma wasseparated from EDTA anticoagulated whole blood by centrifugation at 1000g for 15 minutes and kept in polypropylene tubes. Test plasma wasobtained from recipients prior to transfusion and after any precedingtransfusions whenever possible. In no case was test plasma obtainedafter the transfusion of interest. Positive control plasmas wereobtained from multiply transfused individuals with known high-titerbroad spectrum anti-HLA antibodies, as demonstrated with the immunobeadassay against a panel of normal platelets. Negative control plasmas wereobtained from normal volunteers with no history of transfusion orpregnancy. Both positive and negative control plasmas wereultracentrifuged at 100,000 rpm for 1 hour. All plasmas were used freshor stored frozen at -70° C. Repeated freezing and thawing was avoided.

Sera were prepared from a small number of positive and negative controlplasmas by incubation in glass tubes in the presence of CaCl andthrombin. Complement was inactivated with 0.02 M EDTA.

F. The HLA Immunobead Assay

Aliquots of 10⁸ donor platelets were sensitized by incubation with 100ul of test or control plasma for 1 hour in plastic tubes. The sensitizedplatelets were washed three times, resuspended in 900 ul of citratebuffer, and solubilized by adding 100 ul of 10% Triton X-100. If theassay was to be completed the same day, the lysate was centrifuged at3000 g for 5 minutes and the supernatant transferred to 15 mlpolypropylene tubes. The lysate could be frozen at -20° C. followingsolubilization and the assay completed at a later date without affectingresults.

Immunobeads were prepared by incubating 1/4 inch polystyrene beads(#0023804, Pierce Chemical Co., Rockford, Ill.) with anti-human IgG(HB-43, 10 ug per bead) in 500 ul per bead of 0.01 M NaHCO₃ buffer (pH8.5) for 1 hour on a rotator. After 4 washes with 1.0 ml PBS-Tween theywere incubated for an additional hour with 2% bovine serum albumin (1 mlper bead) to block residual binding sites. Following 4 more washes, 1bead was added to each tube containing platelet lysate and incubatedwith constant agitation for 1 hour. During this incubation, IgGalloantibodies which had been bound to the platelet beforesolubilization attach via the Fc portion to the HB-43 bound to the bead.The class I HLA antigen remains bound to the Fab portion of thealloantibody after solubilization, and is thus bound indirectly to thebead. The beads were washed 4 more times in PBS-Tween, and incubated for1 hour with 400,000 cpm of ¹²⁵ I-labeled monoclonal anti-HLA antibody(HB-95) in 1 ml PBS-Tween. Following 4 final washes, the beads weredried on a paper towel and counted in a Beckman gamma counter. Allincubations were carried out at room temperature.

The assay of interest was run in triplicate along with duplicates of 1positive and 2 negative controls. One technician could run 24simultaneous assays in approximately 6 hours.

G. Radioactive Antiglobulin Test (RAGT)

The RAGT was performed by a modification of the method of LoBuglio etal.²⁷ Briefly, 10⁸ washed platelets were sensitized by incubation with100 ul of plasma for 1 hour at room temperature, then washed three timesin citrate buffer. A 3×10⁷ aliquot was incubated for 30 minutes with300,000 cpm of ¹²⁵ I-labeled anti-human IgG (HB-43). Triplicate 50 ulaliquots containing 6×10⁶ platelets were layered over 200 ul of 30%sucrose in 400 ul plastic microcentrifuge tubes with tapered tips andcentrifuged at 11,000 g for 5 minutes. After quick-freezing at -70° C.,the tips containing the platelet button were removed and counted in aBeck/nan gamma counter.

H. Transfusion Study Results

(i) Characterization of the HLA Immunobead Assay

Triplicate assays using 12 negative control plasmas against the samenormal platelets gave a mean percent variation of 8.7+/-9.0 (1 SD).Absolute values of negative controls generally fell in the range of80-300 cpm. A single negative control plasma was also tested against thesame platelets 10 times, giving a mean per cent variation of 6.3+/-6.6%.Assays run using citrate buffer in place of plasma gave resultscomparable to negative controls.

To assess the possibility that the assay might recognize antiplateletantibodies directed against non-HLA antigens, plasmas from 18 patientswith the clinical syndrome of ITP were tested against normal platelets.Seven of these had plasma autoantibodies to the glycoprotein IIb/IIIacomplex, 2 had autoantibodies to glycoprotein Ib, and the remainder gavenegative results for both these autoantibodies. Three plasmas gavepositive results in the immunobead assay, 2 with GP IIb/IIIaautoantibodies and one with a strong antiplatelet autoantibody withoutdemonstrable specificity. All 3 patients had a history of pregnancyand/or transfusion, suggesting coexisting alloantibodies.

Seven positive and 7 negative controls were run using serum and comparedto results using plasma. No significant difference was noted betweennegative controls. The absolute value of serum positive controlsaveraged 60% of plasma values. No positives were missed by using serum.

(ii) Interpretation of Assay Results

The mean test result was divided by the mean of the negative controls togive a test/control ratio (TCR). Experiments with 12 normals gave a meannormal TCR of 1.00+/-0.14 (1 SD). A TCR of <1.43 was initially definedas negative, to include 3 standard deviations. In practice, TCRs between1.43 and 3.0 were associated with successful transfusion outcomes in 5of 6 cases (Table 5). This was interpreted as evidence that theimmunobead assay can detect levels of alloantibody below that necessaryto significantly affect platelet survival. That this was not due torandom variation is supported by the observation that all 9 A matchtransfusions were associated with TCRs <1.43. For clinical purposes,therefore, a TCR of </=3.0 was considered "negative" and predictive of asuccessful transfusion outcome.

A TCR of </=2.0 was empirically defined as negative for the RAGT bysimilar retrospective data analysis.

                  TABLE 5                                                         ______________________________________                                        TRANSFUSION EPISODES WITH TCRs                                                BETWEEN 1.43 AND 3.00                                                                 Mean Test Mean Control       1 Hour                                   Patient CPM       CPM           TCR  CCI                                      ______________________________________                                        1       344       197           1.75  2659                                    2       390       150           2.61 17396                                    3       123        81           1.53 11481                                    4       380       166           2.29 10298                                    5       505       169           2.99 12269                                    6       266       143           1.86 11538                                    ______________________________________                                         TCR  test/control ratio;                                                      CPM  gamma counts per minute                                             

(iii) Correlation With Transfusion Outcome

Each valuable transfusion episode was categorized according to whetherthe immunobead assay was positive (TCR>3.0) or negative (TCR</=3.0), andthe outcome successful (1 hour CCI >/=7500) or unsuccessful (1 hour CCI<7500). The data for both the immunobead assay and the RAGT aresummarized in Table 6. The immunobead assay was positive in 18 episodes;16/18 (88.9%) had unsuccessful outcomes. In 33 cases the assay wasnegative; 29/33 (87.9%) had successful outcomes. The mean 1 hour CCI was2199 for assay positive episodes, 13072 for assay negative episodes.

                  TABLE 6                                                         ______________________________________                                        CORRELATION OF IMMUNOBEAD AND RAGT                                            RESULTS WITH TRANSFUSION OUTCOME                                              Assay Positive     Assay Negative                                             CCI < 7500   CC > 7500 CCI < 7500 CCI > 7500                                  ______________________________________                                        Im-   16/18 (88.9%)                                                                            2/18      4/33 (12.1%)                                                                           29/33 (87.9%)                             muno-            (11.1%)                                                      bead                                                                          RAFT  15/21 (71.4%)                                                                            6/21      5/30 (16.7%)                                                                           25/30 (83.3%)                                              (28.6%)                                                      ______________________________________                                    

Twenty one episodes were RAGT positive; 15/21 (71.4%) had anunsuccessful outcome. Of the 30 RAGT negative episodes, 25/30 (83.3%)had a successful outcome. The mean 1 hour CCI was 3897 for RAGT positiveepisodes, 11118 for RAGT negative episodes.

In a single instance (involving patient 1), a donor initially associatedwith a positive immunobead assay and an unsuccessful outcome gave anegative assay and a successful outcome 1 month later. The initialtransfusion was given just prior to preparation for BMT with high-dosecyclophosphamide and total nodal irradiation, suggesting that thealloantibody-producing lymphocytes were ablated, followed bydisappearance of preformed antibody.

A plot of TCR vs. 1 hour CCI results from this study demonstrates thedefinition between positive and negative results, with no interveningarea where CCI is proportional to TCR.

(iv) Misclassified Transfusion Episodes

Of the 6 episodes misclassified by the immunobead assay, only 1 (thecase associated with granulocyte transfusion) was correctly classifiedby the RAGT. Six of the 11 episodes misclassified by the RAGT werecorrectly classified by the immunobead assay. In 5 instances, both testsgave misleading results.

In the first immunobead-assay-positive/successful-outcome episode(patient 1), the TCR was 5.3 and the 1 hour CCI 16,897. The 6 hour postplatelet count had fallen to below the pre-transfusion level. The secondcase (patient 6) had a TCR of 253.7 (the 2nd highest recorded during thestudy), a 1 hour CCI of 10806 and a 16 hour CCI of 1965. When the samedonor was used again 4 weeks later, the TCR was 183 and the 8 hour CCI0. Both patients had unsuccessful outcomes with other donors associatedwith a positive immunobead assay.

The 4 assay-negative/unsuccessful-outcome cases were all associated withpotential non-alloantibody-related causes of poor platelet survival. Thefirst (patient 2) was associated with fever and occurred 5 days beforethe patient became refractory to all donors and was dropped from thestudy (see Patient and Donor Population section). The other 3 involvedpatient 1: the first occurred when platelets were given simultaneouslywith a granulocyte transfusion associated febrile reaction, and theother 2 after the patient had been incubated for ARDS but before hebecame refractory to all donors.

(v) Feasibility of Using Stored Platelets

The mean positive control/negative control ratios for both plateletdonors at each time interval are displayed in Table 7. The resultsindicate some deterioration in antigenic integrity or availabilitybetween 1 and 5 days, following which no further major deterioration wasdetectable through the longest interval tested (60 days). Plateletsstored in suspension at 4° C. maintained antigenic integrity slightlybetter than frozen platelets, although the difference was not marked.Although the positive controls used here remained markedly positivethroughout the study, it is conceivable that plasmas with lower titersof anti-HLA antibody might become negative when tested against storedplatelets. There was no significant loss of platelets due to storageduring the study period.

                  TABLE 7                                                         ______________________________________                                        IMMUNOBEAD ASSAY RESULTS USING PLATELETS                                      STORED FROZEN IN-LIQUID NITROGEN                                              AND IN SUSPENSION AT 4° C.                                             Storage      Day of Storage                                                   Method       1       6        15   30     40                                  ______________________________________                                        Platelet Donor #1                                                             4° C. 53.8*   26.7     23.9 25.7   21.0                                liquid N.sub.2                                                                             32.6    20.7     23.9 25.3   10.5                                Platelet Donor #2                                                             4° C. 103.0   33.5     49.3 53.1   48.6                                liquid N.sub.2                                                                             71.5    30.6     35.6 40.5   32.0                                ______________________________________                                         *Data presented as mean positive control/negative control ratios.        

H. Discussion of Transfusion Study Results

In this study, the immunobead assay was adapted to characterizeanti-platelet alloantibodies to Class I HLA antigens and the resultswere applied to the problem of platelet crossmatching. The resultsindicate the test has sufficient predictive value to warrant wider scaleapplication as a predictor of platelet transfusion success. Ideally,prospective donors would have platelets stored when they enter the donorpool. When a patient with a poor response to random donor platelets isencountered, his or her plasma could be assayed against a small panel ofnormal platelets chosen to represent the common HLA antigens. Ifnegative, it could be assumed a problem other than alloimmunizationagainst HLA antigens exists, and the expense of single donor plateletsavoided. If positive, HLA matched platelets would be indicated. In themajority of cases where these are not available, the best matches couldbe pulled from the donor pool and their stored platelets tested,selecting those who test negative. Although our storage study extendedonly 60 days, other studies suggest much longer storage periods arefeasible.

One potential drawback to the immunobead assay is its HLA specificity.Many authors have suggested that alloantibodies to platelet-specificantigens account for some percentage of transfusion failures, usuallybased on poor response to HLA identical platelets and/or the lack ofdemonstrable lymphocytotoxic antibody. However, alloantibodies toplatelet-specific antigens have been difficult to demonstrateconclusively in this setting, and their true significance remainsuncertain. Herman et al. used Western blots to study 7 patientsrefractory to HLA matched platelets, and were unable to demonstrateantibody to platelet specific antigen on the glycoprotein IIb/IIIamolecule. They concluded that antibodies to platelet-specific antigensare not commonly present. Our finding of only 1/51 instances of poortransfusion outcome where the RAGT was positive and the immunobead assaynegative tends to support this. It seems more likely that unrecognizednon-immunologic factors account for many if not all of these cases.However, it would seem prudent at present to use the RAGT or a similarnon-HLA specific assay to evaluate the presence of antibody to otherplatelet-associated antigens in cases where the HLA-specific immunobeadassay is negative in the face of poor platelet survival.

The foregoing is intended as illustrative of the present invention butnot limiting. Numerous variations and modifications may be effectedwithout departing from the true spirit and scope of the invention.

What is claimed is:
 1. A method of assaying platelet compatibilitybetween donor and patient comprising the steps of:(a) admixing analiquot of serum of plasma from a patient with platelets of aprospective donor to form an aqueous, liquid admixture, (b) maintainingsaid admixture under biological assay conditions for a predeterminedtime period sufficient for any alloantibodies present in the serum orplasma to immunoreact with a platelet antigen to form a plateletantigen-alloantibody immunocomplex, (c) lysing the platelets of saidadmixture to form cellular debris and solubilize and plateletantigen-alloantibody immunocomplex, when said alloantibodies and saidplatelet antigen were present in said admixture, (d) separating thecellular debris from any solubilized immunocomplex formed in step (c),(e) affixing any of said separated, solubilized immunocomplex of step(d) present to a solid phase support capable of specifically bindingsaid solubilized immunocomplex to form a solid phase-affixed plateletantigen-alloantibody immunocomplex, and (f) thereafter, assaying for thepresence of said platelet antigen-alloantibody immunocomplex as a solidphase-affixed immunocomplex by reacting the immunocomplex present with alabeled specific binding agent that specifically binds to theimmunocomplex, whereby the absence of immunocomplex indicatescompatibility and the presence of immunocomplex indicatesincompatibility of said donor and patient.
 2. A method of typing cellsfor the presence of at least one of a group of cell surface antigenscomprising the steps of:(a) reacting an aliquot of a cell sample to betyped with antibodies that react with an antigen epitope common tosubstantially all of the group of the cell surface antigens sought toform a common epitope-antibody immunocomplex, (b) lysing the cells ofsaid sample to provide cellular debris and a solubilized commonepitope-antibody immunocomplex, (c) separating the cellular debris fromsaid solubilized immunocomplex formed in step (b), (d) affixing saidimmunocomplex to a solid phase support capable of specifically bindingsaid immunocomplex to form a solid phase-affixed immunocomplex, (e)reacting the solid phase-affixed immunocomplex with labeled paratopicmolecules that immunoreact with at least one specific cell surfaceantigen epitope of the group of cell surface antigens to form a secondsolid phase-affixed immunocomplex, and (f) assaying for the presence ofsaid labeled paratopic molecules in said solid phase-affixed secondimmunocomplex, and thereby for the presence of said at least onespecific cell surface antigen epitope.
 3. The method of claim 2 whereinsaid label comprises a radiolabeling agent.
 4. The method of claim 2wherein said group of cell surface antigens comprises the HLA antigens.5. The method of claim 2 wherein said separated solubilized commonepitope-antibody complex is divided into a plurality of aliquots, saidaliquots are individually reacted with members of a panel of labeledparatopic molecules, and each of said aliquots is then assayed for thepresence of a second immunocomplex.
 6. The method of claim 5 whereinsaid at least one specific cell surface antigen epitope is an epitope ofan HLA antigen.